Abstract Microbubble-based ultrasound contrast agents (UCA) potentiate the effect of sonothrombolysis, without or in combination with tissue Plasminogen Activator (tPA). The underlying mechanisms of why ultrasound (US) accelerates thrombolysis, how to optimize the US settings for effective clot lysis in intracranial arteries and how microbubbles accelerate sonothrombolysis are poorly understood. While we aim to explore and optimize some of these parameters, our goal is to study the impact of US on thrombolysis in combination with tPA and/or UCA microbubbles at settings available on standard diagnostic equipment for transcranial vascular imaging. To achieve this we propose the following Aims: Aim 1 - In Vitro Sonothrombolysis Step 1 A flow system has been established which allows us to study thrombolysis of human whole blood clots exposed to circulating, pulsatile flow and to acquire the acoustic properties at the clot site simultaneously. The experiments planned for Step 1 will allow us to define preferred US parameter settings needed to successfully lyse thrombi and to study the effect of flow on clot lysis when US is applied alone or in combination with tPA and/or UCA. Step 2 Based on what we will learn during Step 1, the three most effective combinations will be repeated to optimize US transmit parameters now insonating through intervening wet human cadaveric temporal skull bone. Insonation through bone is limited by sound absorption, scattering and phase aberration. This experiments will allow us to study these effects and to characterize the acoustic parameters at the clot site during insonation through human cadaveric skull bone. This will help us optimize the US parameters, such as transmit frequency, power, focus etc to achieve transcranial thrombolysis. Aim 2 - In Vivo Sonothrombolysis A rabbit renal artery model has been established. Based on an erythrocyte-rich thrombus model local thrombosis is induced. Using the optimized US parameters determined in Aim 1 we will confirm the contribution of US alone and the combination of US with tPA or UCA or both on accelerating clot lysis in vivo. We believe that the interaction between thrombus and endothelium has an important impact on thrombolysis, which cannot be studied in an in vitro model. The proposed rabbit model will help us study these effects and their impact on thrombolysis in vivo in a system that resembles the middle cerebral artery. More important, it will allow us to asses macro- and microvascular recanalization. The most effective combinations, as determined in Aim 1, will be repeated to optimize the insonation parameters when sound is transmitted through the same wet human cadaveric temporal skull bone used for the in vitro experiments. Although the rabbit brain would be a more ideal organ to study, the rabbit skull will not allow us to translate the knowledge gained to human subjects because of the dramatic differences in skull geometry relative to the US beam. We will instead use the rabbit renal circulation since it presents an arterial bifurcation (aorta to renal) of similar dimensions as the MCA. This model will therefore allow us to study the effect of sonothrombolysis on the macro- and the microcirculation with intervening temporal human skull bone to more closely mimic the human setting. We believe that both aims of this project will contribute to a better understanding whether transcranial diagnostic US can be used for the therapeutic purpose of sonothrombolysis in acute stroke patients. We will learn if tPA activity can be augmented in combination with diagnostic US and if UCA microbubbles further enhance this effect. Moreover, we will learn if transcranial sonothrombolysis can already be achieved with diagnostic US alone and if the combination of diagnostic US plus UCA microbubbles, in absence of tPA, could be a serious therapeutic option in the future. The knowledge gained in this project would facilitate the translation into clinical trials, which is the ultimate goal. Noninvasive 'Image-Guided Sonothrombolysis' at the bedside or even in the ambulance vehicle would be a major achievement in stroke therapy.